Device to lavage a blood vessel

ABSTRACT

The present invention provides for compositions and methods for the preservation of tissues and organs ex vivo and in situ. In addition, the present invention provides for kits that may be used in the preparation of the solutions of the present invention. The present invention also provides a device for perfusing tissues and organs with the solutions of the present invention.

This application is a U.S. national entry of International ApplicationNo. PCT/US01/11834, filed Apr. 10, 2001, which is a continuation-in-partof U.S. application Ser. No. 09/546,860, filed Apr. 10, 2000, now U.S.Pat. No. 6,569,615.

The present invention was financed with government funds. The federalgovernment has certain rights in this invention.

FIELD OF INVENTION

Generally, the present invention relates to the field of tissuepreservation. In particular, the present invention relates to a solutionfor prolonged organ preservation, and more particularly to an aqueoussalt solution for the preservation of grafts prior to transplantation.The invention also provides a method of preserving or maintaining anorgan, comprising contacting the organ with an aqueous salt solution fororgan preservation or maintenance.

BACKGROUND

Many different tissue and organ preservation solutions have beendesigned, as investigators have sought to lengthen the time that atissue or organ may remain extra-corporeally, as well as to maximizefunction of the organ following implantation. Several of the keysolutions that have been used over the years include: 1) the StanfordUniversity solution [see, e.g., Swanson, D. K., et al., Journal of HeartTransplantation, (1988), vol. 7, No. 6, pages 456-467 (mentionscomposition of the Stanford University solution)]; 2) a modified Collinssolution [see, e.g., Maurer, E. J., et al., Transplantation Proceedings,(1990), vol. 22, No. 2, pages 548-550; Swanson, D. K., et al., supra(mention composition of modified Collins solution)]; and 3) theUniversity of Wisconsin solution (Belzer, et al., U.S. Pat. No.4,798,824, issued Jan. 17, 1989). Of those, the University of Wisconsin(UW) solution is currently regarded as the best. (See, e.g., Maurer, E.J., et al., supra).

In addition to the composition of the tissue and organ preservation andmaintenance solution, the method of tissue and organ preservation alsoaffects the success of preservation. Several methods of cardiacpreservation have been studied in numerous publications: 1) warmarrest/cold ischemia; 2) cold arrest/macroperfusion; 3) coldarrest/microperfusion; and 4) cold arrest/cold ischemia. The firstmethod involves arresting the heart with a warm cardioplegic solutionprior to exsanguination and cold preservation, but this method failsbecause of the rapid depletion of myocardial energy stores during thewarm period The second method, which involves arresting the heart with acold preservation solution, is better; but continuous perfusion of theheart with preservation solution during the storage period fails becauseof the generation of toxic oxygen radicals. In addition, the procedureof the second method is cumbersome and does not lend itself to easyclinical use. The third method, first described in the journal Nature in1972 in a system called “trickle perfusion,” is better but alsocumbersome. The fourth method of preservation is that of a coldcardioplegic arrest followed by a period of cold immersion of the heart.The fourth method is currently the standard method of cardiacpreservation. This fourth method reliably preserves hearts for periodsof up to six (6) hours, but less than four (4) hours is considered idealfor this method. Since a longer preservation time is desirable, attemptshave been made to improve preservation solutions in such a way as toreliably preserve hearts and other organs for longer periods of time.

Though the University of Wisconsin (UW) solution is currently theindustry standard of organ preservation solutions, it is limited in thelength of preservation time that it provides. Other solutions have beenproposed (see, for example, U.S. Pat. No. 5,552,267 to Stern), however,these have limited use do to the complicated nature of the composition.

The relationship between the long-term patency and endothelial cellpreservation has been established. Endothelial cells are known to beimportant mediators in regulating platelet, anticoagulant, procoagulant,and fibrinolytic functions. These activities of the endothelium allowfor control of blood flow as well as thrombosis or blood clotting whenthere is endothelial injury. Presently, storage solutions are limited inthe length of storage (up to 125 minutes) and protection provided to theendothelium. This time frame is insufficient depending on the type ofoperation being performed (i.e. whether or not a valve replacement orcarotid endarterectomy will be needed along with bypass) and on thesurgeon performing the operation.

Currently available storage solutions used during bypass surgery varyfrom normal saline, to physiological salt solutions, to heparinizedblood. These solutions do not provide an adequate environment forendothelial or smooth muscle cell support. Normal saline lacks an energysource such as glucose. The pH of saline solutions tend to be low in the6 to 7 range which is hostile to these fragile cells. Heparinized bloodhas only been shown to provide adequate storage of veins only up to 90minutes. All of the currently available solutions are deficient in thecombination of free radical scavengers, antioxidants, and nitric oxidesynthase substrates that can provide a protective environment forcellular support during this time period where much damage occurs.

The saphenous vein is the most commonly used conduit for coronary arterybypass graft (CABG). The intraoperative preservation of harvestedsaphenous veins prior to performance of a CABG is believed to be afactor in the protection of the endothelial cells. Indeed, therelationship between the long-term patency and endothelial cellpreservation is well established. That is to say, the preservation ofendothelial cell viability is vital for inhibiting early pathologicalchanges and the long-term patency of vascular grafts. Zilla P, vonOppell U, Deutsch M. The endothelium: A key to the future. J Card Surg1993;8:32-60. Restenosis of venous bypass grafts, however, is a commonsequelae proximal to vascular endothelial injury that occurs during veinharvest.

The pathological changes leading to ultimate vein graft occlusion andloss in vasomotor function are well documented. Verrier E D, Boyle E M.Endothelial cell injury in cardiovascular surgery: An overview. AnnThorac Surg 1997;64:S2-8. Endothelial damage appears to be a major causeof graft failure. Specifically, this injury may occur at the time ofharvest due to blunt surgical trauma and stretch and due to distensionor pressurization prior to anastomosis. Endothelial trauma is alsocaused by exposure to arterial circulation pressure and oxygenated bloodafter graft insertion.

Additionally, endothelium damaged or denuded saphenous veins are highlysensitive to the very potent, endothelium derived, circulatingendogenous vasoconstrictors. For example, endothelin-1, TXA₂, andangiotensin II known to increase during CABG surgery. The increase invascular tone mediated by these vasoconstrictors may lead to attenuatedblood flow, stasis, and predisposition to thrombus formation in venousgrafts.

What is needed is a physiological salt solution that would prolong thestorage and protection available to harvested bypass conduits and otherorgans such as those used for transplantation in excess of 24 hours onthe basis of cell viability and the integrity of key cell regulatorypathways, including nitric oxide synthesis. In addition devices andmethods are also needed that precondition blood vessel grafts withoutdamaging the endothelium of said blood vessels during perioperativegraft preparation.

SUMMARY OF INVENTION

Generally, the present invention relates to the field of tissuepreservation. In particular, the present invention relates to a solutionfor prolonged organ preservation, and more particularly to an aqueoussalt solution for the preservation of grafts prior to transplantation.The invention also provides a method of preserving or maintaining anorgan, comprising contacting the organ with as aqueous salt solution fororgan preservation or maintenance.

Adequate preservation of organs intended for transplantation is criticalto the proper functioning of the organ following implantation. Thisinvention concerns an organ preservation or maintenance solution thatcan preserve organs intended for transplantation for periods of timethat are longer than the currently best solution available. Inparticular, the present invention concerns the preservation of venousand arterial grafts. A longer preservation time is desired to enablecross-matching of donor and recipient to improve subsequent survival, aswell as to allow for coast to coast and international transportation oforgans to expand the donor and recipient pools. Experimental work forthis invention has focused on the heart and heart tissues, but the organpreservation or maintenance solution of the subject invention may beused for other organs, and for tissues and cells, as well.

The organ preservation or maintenance solution of the present inventionshows a substantial improvement over the prior art for increasing thepreservation time for organs intended for transplantation. (SeeExperimental section). The organ preservation or maintenance solution ofthis subject invention shall be referred to as the GALA solution (namedafter Glutathione, Ascorbic acid, L-Arginine).

The present invention differs from other organ preservation solutions ofthe prior art in a number of respects. In our experiments, none of thesesolutions were able to preserve the structural integrity and function ofsaphenous vein endothelium for more than 2 hours. The present inventionincludes NOS substrates and antioxidants and is simple to prepare, beingcomposed of a limited number of ingredients. Additionally, it does notrequire the elimination of sodium, calcium and chloride from thesolution, as does at least one prior art solution (see U.S. Pat. No.5,552,267 to Stem, et al.). In these regards, the present invention isimproved over prior art compositions in that it permits the viability oftissue to be maintained longer than in traditional solutions and it iseasier to prepare.

The GALA solution of the present invention is based on Hank's balancedsaline solution. Hank's balanced salt solution (HBSS) is a commerciallyavailable physiological salt solution containing D-glucose 1 g/L,calcium chloride (anhydrous) 0.14 g/l, potassium chloride 0.4 g/l,potassium phosphate 0.06 g/l, magnesium chloride.6H₂O 0.1 g/l, magnesiumchloride.7H₂O 0.1 g/l, sodium chloride 8 g/l, sodium bicarbonate 0.35g/l, and sodium phosphate 0.048 g/l. The present invention modifies HBSSby the addition of ascorbic acid (vitamin C), reduced glutathione,L-arginine, and heparin to a final concentrations of about 500 μM, 1000μM, 500 μM, and 50 Units/ml, respectively. The pH is then adjusted to7.4 using 10 M sodium hydroxide. To date, no known preservation solutionfor harvested veins and arteries has been enhanced with ascorbic acid,glutathione, L-arginine, and heparin in an attempt to preventendothelial injury. This new solution provides free radical scavengers,antioxidants, an NO substrate, a reducing agent, an energy source(glucose), an anti-coagulant, and physiological concentrations ofelectrolytes and buffers. As demonstrated in the Experimental section(below), the solution has the unexpected benefit of providing a greatlyextended preservation time over the available prior art preservationsolutions.

The present invention is not limited to the compositions listed above.Adenosine may be added as a supplemental energy source. Adenosine may beadded a concentration of about 500 μM-5000 μM. Additionally, Lacidipine,a vasorelaxant calcium channel blocker, may be added to GALA in thefinal concentration of about 1 pM-1 mM. Additionally still, vasoactiveintestinal peptide (VIP) may be added to GALA in the final concentrationof about 1 μM-1 mM. Additionally still, Endothelin receptoragonists/antagonists (ETa and Ebb-receptors) may be added to GALA.Although the present invention is not limited to any particularmechanism, endothelin receptor agonists/antagonists work asvasocontractors and vasorelaxants, respectively. Furthermore, ananticoagulant need not be added, for example, in situations where thetissue or organ has been perfused of blood. Further still, glutathioneneed not be added because, for example, it is partly synergistic withascorbic acid. Therefore, it is contemplated that a minimal formulationof the present invention would be HBSS with ascorbic acid and L-arginineadded in to the concentrations listed above.

The present invention is not limited to any particular concentration ofthe ingredients listed above. In one embodiment, the concentration ofascorbic acid is between about 25-1000 μM. In another embodiment, theconcentration of glutathione is between about 50-2000 μM. In yet anotherembodiment, the concentration of L-arginine is between about 250-20001μM. In still yet another embodiment, the concentration of heparin isbetween about 50-250 units/l. The present invention is not limited toany particular pH. In one embodiment the pH of the solution is betweenabout pH 6.6-8.0. More preferably, the pH is between about pH 7.0-7.6.The present invention is not limited to any particular anticoagulant. Inone embodiment, the anticoagulant is heparin. In another embodiment theanticoagulant is hirudin. The solution of the present invention maycontain certain bacteriostats. The bacteriostat may be selected from agroup comprising penicillin and cerfazolin. Other bacteriostats may beused. Selection of a bacteriostat may be determined at the time ofpracticing the invention. For example, allergies may be taken intoaccount when selecting a bacteriostat.

The solutions, devices, and perfusion methods of the present inventionare not limited to use with a particular tissue, organ or cell type. Forexample, the invention may be used with harvested saphenous veins,epigastric arteries, gastroepiploic arteries and radial arteries used incoronary bypass grafting (CABG). The present invention may also be usedto maintain organs and tissue during transplant operations. The presentinvention is not limited to any particular tissue or organ. For example,it is contemplated that such organs or tissues may be heart, lungs,kidney, brain, muscle grafts, skin, intestine, bone, appendages, eyes,etc or portions thereof. Additionally, the present invention may be usedas an in situ tissue or organ preservative. It is contemplated that thesolution of the present invention be used to wash and bath tissues andorgans that have not been removed from the patient. For example, it iscontemplated that the present invention be used during cardioplegia. Itis also contemplated that the present invention be used in, for example,emergency procedures where a tissue or organ may need to be bathed topreserve it until surgery or other medical attention can be obtained. Inthis regard, the solution may be made available to emergency medicalpersonnel both in hospital settings and “in the field” (i.e., inambulances or in temporary emergency medical facilities).

The present invention contemplates the present invention may be anaqueous solution or the present invention may be composed of powders andconcentrated solutions that could be mixed with sterile water, asneeded. The present invention also contemplates that the invention maybe composed of a quantity of HBSS along with a supplement package thatmay be mixed with the HBSS.

The present invention contemplates an aqueous solution for organ andtissue preservation, comprising: a) calcium ions; b) D-glucose (fromabout 50 mM to about 120 mM); c) potassium ions (from about 100 mM toabout 250 mM; derived from compounds selected from the group consistingof potassium chloride, and potassium phosphate); d) magnesium ions (fromabout 2 mM to about 20 mM; derived from compounds selected from thegroup consisting of magnesium sulfate, and magnesium chloride); e)sodium ions; f) ascorbic acid in a concentration of about 25-1000 μM; g)glutathione in a concentration of about 50-2000 μM; h) L-arginine in aconcentration of about 250-2000 μM; i) an anticoagulant (selected fromheparin and hirudin) at a concentration sufficient to substantiallyinhibit blood coagulation (for heparin this would be from about 50units/l to about 250 units/l); and j) a buffer (the buffer is selectedfrom the group consisting of sodium phosphate and sodium bicarbonate) inan amount sufficient to maintain the pH of said aqueous organpreservation solution at about 6.8 to 8.0.

The present invention contemplates an aqueous solution for organ andtissue preservation, comprising: a) calcium ions; b) D-glucose (fromabout 50 mM to about 120 mM); c) potassium ions (from about 100 mM toabout 250 mM; derived from compounds selected from the group consistingof potassium chloride, and potassium phosphate); d) magnesium ions (fromabout 2 mM to about 20 mM; derived from compounds selected from thegroup consisting of magnesium sulfate, and magnesium chloride); e)sodium ions; f) ascorbic acid in a concentration of about 25-1000 μM;

-   -   g) glutathione in a concentration of about 50-2000 μM; h)        L-arginine in a concentration of about 250-2000 μM; i) an        anticoagulant (selected from heparin and hirudin) at a        concentration sufficient to substantially inhibit blood        coagulation (for heparin this would be from about 50 units/l to        about 250 units/l); and j) a buffer (the buffer is selected from        the group consisting of sodium phosphate and sodium bicarbonate)        in an amount sufficient to maintain the pH of said aqueous organ        preservation solution at about 6.8 to 8.0; and k) tissue.        Additionally, the present invention contemplates that the tissue        is saphenous vein.

The present invention contemplates a method for preserving tissuecomprising:

-   -   i. providing a tissue; ii. contacting said tissue with a        solution comprising: a) calcium ions in an amount sufficient to        support intracellular function and maintenance of cellular        bioenergetics; b) D-glucose in an amount sufficient to support        intracellular function and maintenance of cellular        bioenergetics; c) potassium ions in an amount sufficient to        support intracellular function and maintenance of cellular        bioenergetics; d) magnesium ions in an amount sufficient to        support intracellular function and maintenance of cellular        bioenergetics; e) sodium ions in an amount sufficient to support        intracellular function and maintenance of cellular        bioenergetics; f) ascorbic acid in a concentration of about        25-1000 μM; g) glutathione in a concentration of about 50-2000        μM; h) L-arginine in a concentration of about 250-2000 μM; i) an        anticoagulant at a concentration sufficient to substantially        inhibit blood coagulation; and f) a buffer in an amount        sufficient to maintain the average pH of said aqueous organ        preservation solution at about a physiological ph or above.

Additionally, the present invention contemplates a kit for thepreparation of an organ preservation solution comprising: i. acontainer; ii. an aqueous solution disposed within said containerwherein said solution comprises; a) D-glucose; b) calcium chloride; c)potassium chloride; d) potassium phosphate; e) magnesium chloride 6.H₂O;f) magnesium chloride 7.H₂O; g) sodium chloride; h) sodium bicarbonate;and i) sodium phosphate; iii. a supplement for introduction into saidsolution comprising; j) ascorbic acid; k) glutathione; 1) L-arginine;and m) heparin.

Furthermore, the present invention contemplates a kit for thepreparation of an organ preservation solution comprising: i. acontainer; ii. reagents deposited in said container, said reagentscomprising, a) D-glucose; b) calcium chloride; c) potassium chloride; d)potassium phosphate; e) magnesium chloride 6.H₂O; f) magnesium chloride7.H₂O; g) sodium chloride; h) sodium bicarbonate; and i) sodiumphosphate; j) ascorbic acid; k) glutathione; l) L-arginine; m) heparin;and n) sterile water.

The present invention contemplates a composition comprising an aqueoussalt solution comprising an antioxidant, glutathione, an L-amino acidand an anticoagulant. Additionally, the present invention contemplatesthe composition wherein it also comprises isolated tissue. Particularly,the isolated tissue may be a vein, and more particularly, a saphenousvein.

The composition of the present invention may also comprise glucose.Furthermore, the antioxidant of the present invention may be ascorbicacid. Yet further still, the concentration of the ascorbic acid is about25-1000 μM. Even further still, the concentration of the glutathione isof about 50-2000 u. Even further still, the L-amino acid is L-arginine.Even further still, the L-arginine is present in a concentration ofabout 250-2000 u. Even further still, the anticoagulant is selected fromthe group consisting of heparin and hirudin. Even further still, theanticoagulant is heparin, and wherein said heparin is present in aconcentration of between about 50 units/ml and about 250 units/ml.

The present invention contemplates a composition comprising an isolatedtissue in an aqueous salt solution comprising an antioxidant,glutathione, an L-amino acid and an anticoagulant. Further still, thetissue is an isolated vein. Even further still, the vein is a saphenousvein. Even further still, the antioxidant is ascorbic acid and theascorbic acid is present in a concentration of about 25-1000 u. Evenfurther still, the glutathione is present in a concentration of about50-2000 u. Even further still, the L-amino acid is L-arginine and ispresent in a concentration of about 250-2000 u. Even further still, theanticoagulant is selected from the group consisting of heparin andhirudin. Even further still, the anticoagulant is heparin, and whereinsaid heparin is present in a concentration of between about 50 units/mland about 250 units/ml.

The present invention contemplates a composition comprising an isolatedhuman tissue in an aqueous salt solution comprising an antioxidant,glutathione, an L-amino acid and an anticoagulant. Further still, thetissue is an isolated human vein. Even further still, the vein is asaphenous vein. Even further still, the antioxidant is ascorbic acid andthe ascorbic acid is present in a concentration of about 25-1000 u. Evenfurther still, the glutathione is present in a concentration of about50-2000 u. Even further still, the L-amino acid is L-arginine and ispresent in a concentration of about 250-2000 u. Even further still, theanticoagulant is selected from the group consisting of heparin andhirudin. Even further still, the anticoagulant is heparin, and whereinsaid heparin is present in a concentration of between about 50 units/mland about 250 units/ml.

The present invention contemplates a method, comprising: a) providing i)an isolated tissue and ii) an aqueous salt solution comprising anantioxidant, glutathione, an L-amino acid and an anticoagulant; and b)contacting said isolated tissue with said aqueous salt solution.Furthermore, the tissue is an isolated vein. Even further still, the isa saphenous vein. Even further still, the antioxidant is ascorbic acidand the ascorbic acid is present in a concentration of about 25-1000 u.Even further still, the glutathione is present in a concentration ofabout 50-2000 u. Even further still, the L-amino acid is L-arginine andis present in a concentration of about 250-2000 u. Even further still,the anticoagulant is selected from the group consisting of heparin andhirudin. Even further still, the anticoagulant is heparin, and whereinsaid heparin is present in a concentration of between about 50 units/mLand about 250 units/mL.

The present invention also contemplates devices and methods forperfusing tissues and organs, and in particular blood vessels andportions thereof. In one embodiment, the present invention contemplatesa device, said device comprising, an incomplete, hollow circuit defininga liquid flow path in fluidic communication with a chamber, saidincomplete circuit comprising a pump operably linked to pressuretransducer (or sensor), said circuit terminating at first and secondattachment points, said first and said second attachment pointsconfigured to accept a (substantially hollow) blood vessel (or segmentthereof) having first and second ends, such that attachment of saidfirst end of said blood vessel to said first attachment point and saidsecond end of said blood vessel to said second attachment pointgenerates a complete, hollow circuit defining a liquid flow path(thereby permitting the circulation of liquid through said circuit andsaid vessel).

It is not intended that the present invention be limited to the precisedesign of the first and second attachment points. It is only importantthat they be designed so as to permit attachment of tissues, such asvessels and segments thereof. In one embodiment, said first attachmentpoint comprises a serrated nozzle. In a preferred embodiment, secondattachment point comprises an adjustable serrated nozzle.

Since the present invention contemplates using human tissue inconnection with said device, it is preferred that said chamber issterile. Moreover, to avoid contamination, it is preferred that saidchamber is disposable.

It is not intended that the present invention be limited to the natureof the material from which the hollow circuit of the device isfabricated. In one embodiment, said incomplete, hollow circuit isfabricated from a polymer. A convenient source of material for thecircuit is tubing.

A variety of configurations for the circuit are possible and the presentinvention is not limited to circular flow paths. To be a “circuit” it isonly necessary that a flow path for fluid be defined such that acomplete traversal of which without local change of direction requiresreturning to the starting point. Of course, where the circuit is“imcomplete,” it is not possible to return to the starting point;however, where the circuit is “complete” (e.g. completed by theattachment of a vessel or segment thereof), at least a portion of theliquid traversing the circuit will return to the starting point on theflow path. A “complete” circuit is largely “closed” in order to preservethe amount or level of liquid in the circuit. However, the presentinvention permits the circuit to be in liquid communication with both areservoir and a chamber. The reservoir provides a source of liquid (e.g.aqueous salt solution). The chamber, when filled with liquid, allows forthe “bathing” of the tissue or vessel on the outside, while the tissueor vessel (through the connection to the circuit) is perfused on theinside. Where tubing is used, said tubing may terminate either outside,inside or at the edge of said chamber, so as to define said first andsecond attachment points.

In an alternative embodiment, the device can comprise a “complete”circuit wherein a segment of the circuit (e.g. a segment of the tubing)is removable. In such an embodiment, the removable segment is removedand replaced with the tissue or vessel. In yet another embodiment, thecircuit is “complete” and comprises extension points which allows forthe attachment of a vessel (or portion thereof) so as to extend thecircuit (or create a second circuit). In such an embodiment, liquid cancirculate through the complete circuit or through the extended (orsecond) circuit.

The device of the present invention can be used with a variety oftissues and methods. In one embodiment, the method comprises a)providing i) a device comprising, an incomplete, hollow circuit defininga liquid flow path in fluidic communication with a chamber, saidincomplete circuit comprising a pump operably linked to pressuretransducer (or sensor), said circuit terminating at first and secondattachment points, said first and said second attachment pointsconfigured to accept a blood vessel having first and second ends; andii) a segment of a blood vessel, said blood vessel having a first endand a second end; and b) attaching, in any order, said first end of saidsegment to said first attachment point and said second end of saidsegment to said second attachment point, under conditions such that acomplete, hollow circuit defining a liquid flow path is produced. In apreferred embodiment, the method further comprises, after step b,circulating an aqueous solution in said hollow circuit. In such anembodiment, a variety of solutions can be used; however, in a preferredembodiment, said aqueous solution comprises an antioxidant andL-arginine and may (optionally) further comprises an anticoagulant. anda cellular reducing agent.

The methods and devices described above can be used with a variety oftissues, organs and vessels. In one embodiment, a blood vessel is used,such as an isolated vein (e.g. a saphenous vein)

DESCRIPTION OF FIGURES

FIG. 1 shows the viability of human saphenous vein stored in variousstorage solutions.

FIG. 2 shows the effects on endothelial nitric oxide synthase (egos)activity after 5 hours of storage in HBSS v. GALA solution.

FIG. 3 shows a comparison of cell viability of human saphenous veinsstored in HBSS verses GALA solution.

FIG. 4 shows cell viability of a human saphenous vein following 24 hoursof storage in GALA solution.

FIG. 5 shows immunofluorescence labeling of SVG stored in GALA and HLSfor 3 hours.

FIG. 6 shows one embodiment of a perfusion device contemplated by thepresent invention.

FIG. 7 shows an alternative configuration of the perfusion devicedepicted in FIG. 6.

FIG. 8 is a bar graph showing data comparing physiological functionbetween human saphenous vein grafts distended according to methodsdescribed in the prior art and veins distended according to perfusionmethods of the present invention (as compared to undistended control).

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. For purposes of the present invention, thefollowing terms are defined below.

As used herein, the term “patient” includes members of the animalkingdom including but not limited to human beings.

As employed herein, “organ” includes, but is not limited to, the heart,veins, arteries, lungs, liver, pancreas and the kidneys. Portions oforgans are also contemplated.

As used herein, “sterile water” includes, but is not limited to, (a)sterile water for injection, USP, (b) sterile distilled deionized water,and (c) sterile water for irrigation.

As used herein, “cardioplegia” includes, but is not limited to,paralysis of the heart.

As used herein, “moderate hypothermia” is about 10°-21° C.

As used herein, an “antioxidant” is a substance that, when present in amixture or structure containing an oxidizable substrate biologicalmolecule, delays or prevents oxidation of the substrate biologicalmolecule. For example, ascorbic acid is an antioxidant.

“Balanced salt solution” is defined as an aqueous solution that isosmotically balanced to prevent acute cell or tissue damage.

“Buffered salt solution” is defined as a balanced salt solution to whichchemicals have been added to maintain a predetermined physiological pHrange.

“Graft” is defined as tissue that is transplanted or implanted in a partof the body to repair a defect.

“Harvested bypass conduit” is defined as a surgically installedalternate route for the blood to bypass an obstruction.

“Solution of cardioplegia” is defined as a solution that aids in thepreservation of the heart during transport or surgery.

“Cellular reducing agent” is defined as an a substance that loseselectrons easily thereby causing other substances to be reducedchemically.

“Physiological solution” is defined as an aqueous salt solution which iscompatible with normal tissue, by virtue of being isotonic with normalinterstitial fluid.

DESCRIPTION OF DETAILED EMBODIMENTS

Generally, the present invention relates to the field of tissuepreservation. In particular, the present invention relates to a solutionfor prolonged organ preservation, and more particularly to an aqueoussalt solution for the preservation of grafts prior to transplantation.The invention also provides a method of preserving or maintaining anorgan, comprising contacting the organ with as aqueous salt solution fororgan preservation or maintenance. As such, the present invention is anovel solution that greatly increases the length of time the tissue ororgan may remain out of the body. In addition, the present inventionalso presents a perfusion apparatus and methods of perfusion, whichreduce endothelial damage to harvested tissues.

The organ preservation or maintenance solution may further comprise areducing agent in an amount sufficient to help decrease reperfusioninjury secondary to oxygen free radicals. The role of glutathione as acellular reducing agent and L-arginine as the substrate for nitric oxidesynthase has been well established. Studies have shown that the oraladministration of a glutathione substrate improved endothelial dependentblood flow in patients with coronary artery disease.

The organ preservation or maintenance solution may further comprise anantioxidant in an amount sufficient to help decrease reperfusion injurysecondary to oxygen free radicals. The antioxidant is selected from thegroup consisting of butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), Vitamin C (ascorbic acid), Vitamin E, or suitablecombinations thereof. Other suitable antioxidants may be used. In apreferred embodiment, the antioxidant is butylated hydroxyanisole (BHA)at a concentration range from about 25 microM to about 100 microM, aloneor in combination with butylated hydroxytoluene (BHT) at a concentrationrange from about 25 microM to about 100 microM.

Currently, the protective role of ascorbic acid on endothelial cells isbeing investigated. Ascorbic acid is known to reduce platelet activationand leukocyte adhesion which are important events in the development ofatherosclerosis. Ascorbic acid is also thought to contribute to thereduction in smooth muscle proliferation which is a key component ofvein graft failure. Work by Jones and colleagues examined the protectiveeffect of ascorbic acid by showing decreased adherence of neutrophils tothe endothelium and free radical scavenging in human umbilical veinendothelial cells (HUVEC). Utoguchi and colleagues showed a decrease inendothelial layer permeability via ascorbic acid-mediated collagensynthesis in HUVEC monolayers. Adams and colleagues observed similarreductions in neutrophil-endothelial cell interactions after oraladministration of L-arginine in cigarette smokers.

The organ preservation or maintenance solution may further comprise ananticoagulant in an amount sufficient to help prevent clotting of bloodwithin the capillary bed of the organ. The anticoagulant is selectedfrom the group consisting of heparin or hirudin. Other suitableanticoagulants may be used. In a preferred embodiment, the concentrationof heparin ranges from about 50 units/l to about 250 units/l.

Anticoagulants are believed to help in preventing clotting of bloodwithin the capillary bed of the preserved organ. Specifically,anticoagulants are believed to help prevent a total organ no-reflowphenomenon at the level of the microcirculation, which would beundesirable following re-implantation and could result in graft failure.Anticoagulants are believed to be helpful in ensuring that thrombosisdoes not occur during or after preservation, so that nutrient deliveryand toxin removal can proceed.

The present invention contemplates a solution for tissue and organpreservation that is superior to prior art solutions in both the lengthof time it is able to preserve the tissue or organ, and in its ease ofpreparation. In one embodiment, the solution is based on HBSS. However,other solutions may be utilized as the basis of the tissue and organpreservation solution of the present invention. Any balanced salinesolution could be used. For example, phosphate buffered saline, Ringer'ssolution, culture medias and cardiopelgic solutions could be used.

The present invention may be used as a cardioplegic solution. In thisregard, the present solution would be administered to the patient'schest cavity after the heart was paralyzed by, for example, an injectionof a potassium enriched solution. After the heart has depolarized, thechest cavity would be flooded with the solution of the presentinvention, or with an available cardioplegic solution with addedglutathione, L-arginine, heparin and ascorbic acid. Additionally, thepotassium concentration would be monitored and supplemented as necessaryto maintain cardioplegia In another embodiment, the present inventionprovides a solution for cardioplegia during cardiac surgery and asolution for preserving a patient's heart for transplantation.Cardioplegia involves arresting the patient's heart or harvesting thepatient's organ, perfusing the heart or organ with an aqueous solutionof the present invention, and removing at least a substantial portion ofthe solution from the heart or organ to effect the removal of wasteproducts from the heart or organ. Additionally, the present may be usedfor tissue and organ preservation for organs that are still within thepatient's body or for tissues and organs that have been removed (forexample, for transplantation). In this regard, the present invention maybe used as a storage solution after removal of the tissue or organ or asa transportation solution for organs that need to be transferred to anew location for the transplantation to take place (for example, fortransport of a heart, kidney or liver from a donor to a recipient). Thepresent invention makes the transport of organs over much longerdistances than can presently be accomplished.

Experimental

Suppliers

Live-Dead assay kit (calcein AM/ethidium homodimer) was obtained fromMolecular Probes, Eugen, Oreg. Membrane permeable 4,5-diaminofluoresceindiacetate (DAF-2/DA) was purchased from Calbiochem, La Jolla, Calif.Hank's balanced salt solution (HBSS), Minimum essential medium (MEM) andRPMI 1640 medium were obtained from GibcoBRL, Grand Island, N.Y. HLSsolution (heparin, 40 units/ml; lidocain, 0.0016%; saline, 0.9% NaCl)was from the Clinical Pharmacy at VA Medical Center, West Roxbury, Mass.L-arginine, reduced glutathione, L-ascorbic acid, bradykinin andN^(w)-nitro-L-arginine (LNNA) were purchased from Sigma Chemical Co.,St. Louis, Mo. Specially designed chambers used for inverted microscopyconsisting of a sterile 35 mm petri plate with a No. 1.5 coverslipsealed over a 10 mm hole in the bottom of the petri plate were obtainedfrom MetTek Corp., Ashland, Mass. Pumps suited to the claimed perfusiondevice include, but are not limited to, Perimatic programmableperistaltic pump, catlog no. HX20722. Varistaltic Pump/Dispenser,catalog no. HX 20721. Fisherbrand-Variable-Flow Peristaltic pumps,catalog no. 13-876-2. Watson-Marlow PumpPro Variable speed transferpump, catalog no. 144-283-2. Tubing suited to the perfusion circuitdescribed in the present invention may be obtained from FisherScientific and Watson-Marlow.

EXAMPLE 1

Hank's balanced salt solution (HBSS), a commercially availablephysiological salt solution (Gibco/BRL, Grand Island, N.Y.) containingD-glucose 1 g/L, calcium chloride (anhydrous) 0.14 g/l, potassiumchloride 0.4 g/l, potassium phosphate 0.06 g/l, magnesium chloride.6H₂O0.1 g/l, magnesium chloride.7H₂O 0.1 g/l sodium chloride 8 g/l, sodiumbicarbonate 0.35 g/l, and sodium phosphate 0.048 g/l was modified by theaddition of ascorbic acid (vitamin C), reduced glutathione, L-arginine,and heparin to a final concentration of 500 μM, 1000 μM, 500 μM, and 50Units/ml, respectively. The pH was then adjusted to 7.4 using 10 Msodium hydroxide. To date, no known preservation solution for harvestedveins and arteries has been enhanced with ascorbic acid, glutathione,L-arginine, and heparin in an attempt to prevent endothelial injury.This new solution (known as GALA solution named after Glutathione,Ascorbic acid, L-Arginine) provides free radical scavengers,antioxidants, an NO substrate, a reducing agent, an energy source(glucose), an anti-coagulant, and physiological concentrations ofelectrolytes and buffers.

After approval from the Human Studies Subcommittee, discarded segmentsof saphenous veins used as bypass conduits were obtained from theoperating room and transported to the laboratory for experimental work.Human saphenous veins (SVG) were excised and obtained from male patientsundergoing cardiac bypass surgery at the West Roxbury Va. MedicalCenter, according to the protocol established by the scientificevaluation committee at the VA Medical Center. A 100 mm segment of 11.5mm diameter was excised from the SVG or its branch in the operating roomand immediately transferred to a storage solution maintained at 21° C.The vessel was transported to the Multi-photon microscopy laboratory inthe Imaging Core facility of the West Roxbury Va. Medical Center forfurther processing, within 10 min after removal of the vessel from thepatient. The vessel was minimally disturbed to protect it from blunttrauma during handling and processing. Excess adipose and adventitiawere gently excised and the vessel was washed of any excess blood withthe storage solutions under investigation. Prior to the assays, 8 mmsegment of the vessel was cut as required and immediately transferred tothe assay solution for further incubation. The rest of the vessel wasmaintained at 21° C. in the assay solution under similar conditions asthose utilized in the OR during bypass surgery. In the initial set ofexperiments, four conventional types of storage solutions were studiedfor their protective effects on the saphenous veins in short-termstorage. Vein segments were initially exposed to 60, 90, and 120 minutesof storage at 21° C. in either: 1) heparin-lidocaine-saline (HLSsolution) containing 200 ml of 0.9% saline, 50 units/ml of heparin, and40 ml of 1% lidocaine, 2) autologous blood with heparin 50 U/ml, 3) HBSSwith heparin 50 U/ml or 4) RPMI/M199 culture medium in a 1:1 mixturewith heparin 50 U/ml added.

To study the protective effects of GALA solution during prolongedstorage conditions, experiments were performed using segments ofsaphenous vein exposed to 1-5 hours of at room temperature (21° C.).Control experiments were performed using HBSS as the storage solutionfor the same 5-hour exposures. To study the effects of overnightstorage, vein segments were exposed to an additional 19 hours of storageat 4° C. in GALA solution prior to performance of viability andfunctionality assays.

Cell viability was measured using calcein-dependent green fluorescenceand cell death was measured using ethidium homodimer-mediated redfluorescence. Following the appropriate exposures to the storagesolutions, the veins were then incubated in a 15 μM solution ofcalcein/ethidium homodimer for 30 minutes at 21° C. for loading of thefluorophores. Imaging of vein segments was performed using the BioRadMRC 1024 ES multi-photon (MPM) imaging system coupled with a mode-lockedSpectra-Physics tunable Tsunami titanium/sapphire laser system tuned to790 nm (pulse duration<80 fs, repetition rate 82 MHz) and a ZeissAxiovert S100 inverted microscope equipped with a high-quality immersion40×/1.2 NA objective. The structural/functional viability of thesaphenous veins was measured at 40× and/or 80×(zoom) magnification. Thelumen and endothelial cell layers were identified by XYZ scanning atdepths ranging from 30-200 μm. Data was expressed as percentage ofliving to dead cells. A viability score was derived based on thepercentage of viable cells to non-viable cells and assigned for eachstorage solution.

Functionality of the veins was assessed by measuring the generation ofNO induced by bradykinin (10 μM) activation of endothelial nitric oxidesynthase (egos), the enzyme responsible for NO synthesis fromL-arginine. The exposures to the storage solutions were performed in thesame manner as in the viability studies at room temperature, and anadditional 19 hours of storage at 4° C. The endothelial cells werelabeled by incubating with 15 μM diaminoflurescein (DAF) in HBSS for 60min at 37° C. DAF is a cell permeable NO fluorophore that fluorescesupon reaction with intracellularly-generated NO. The temporal increasein DAF fluorescence due to generation of NO was measured for 20 minutesusing quantitative MPM imaging and BioRad LaserSharp software. Inquantitating relative NO fluorescence, boundaries were drawn along theendothelial layer in 2 or 3 regions of the vessel lumens. Changes in theintegrated fluorescence intensities over all pixels within theboundaries of each region were measured. The morphology of the regionvaried in terms of size and shape. As a result, the fluorescenceintensities quantitated from each image were normalized to the referenceimage recorded prior to bradykinin activation. The data were expressedas a change in relative fluorescence intensity in reference to thepre-bradykinin non-stimulated image.

As a measure of endothelial cell functionality, egos activity wasquantitated using bradykinin as the enzyme agonist. FIG. 1 depicts theresults after storing vein segments in either Hank's balanced saltsolution (HBSS), heparin-lidocaine-saline solution (HLS), heparinizedblood, or a 1:1 mixture of RPMI and M199 culture media. A markeddecrease in egos activity was noted following 90 and 120 minutes ofstorage. HBSS was superior to all other solutions in the initial set ofexperiments.

EXAMPLE 2

We next sought to improve further on the composition of HBSS by addingcompounds with theoretical value in sustaining endothelial cellviability and cellular function. The end result was the GALA solution.An evaluation of the ability of HBSS and GALA solution to protect theendothelial cells during prolonged storage was then undertaken. Theother three types of storage media were not tested under these prolongedconditions due to the lack of predicted egos activity and viability.FIG. 2 shows the effects on egos activity after 5 hours of storage inHBSS v. GALA solution. Very little activity was noted in cells storedfor 3, 4, and 5-hour exposures as compared to the 1 and 2 hour exposuresfor HBSS. In marked contrast, egos activity is sustained even afterstorage in GALA solution for up to 5 hours. The addition of ascorbicacid, glutathione, and L-arginine substantially prolonged the protectionand functionality of the endothelial cells compared to the conventionalHBSS solution (P<0.001 for egos activity of HBSS v. GALA for the 2, 3,4, and 5-hour exposures).

Endothelial cell viability was also measured for the various storagesolutions. Cells and tissues were observed as follows. BioRad MRC 1024ESmulti-photon imaging system was coupled with a mode-lockedSpectra-Physics tunable Tsunami Titanium/Sapphire laser system tuned to790 nm, (pulse duration <80 fs, repetition rate 82 MH) and a ZeissAxiovert S100 inverted microscope equipped with a high quality waterimmersion 40×/1.2 NA, C-apochroma objective was used to image the SVG at40× and/or 80×(Zoom) magnification. The vessel lumen and the endothelialcell layer were identified by XYZ scanning and were generally at depthsof 100-200 μ depending on the size of the vessel. The scan parametersare chosen such that 512×512 pixel images are generated with pixelresidence time of 0.413 μsec. The images were reconstructed using theBioRad Laser Sharp software.

Table 1 lists the viability scores of the five storage solutionsexamined. In the Live-Dead assay, cells were considered living and/ordead when green and/or red fluorescence was observed, respectively. Thevessel endothelium viability results were quantitatively expressed on ascale of 1-4. 4+ indicates a structurally intact endothelial layer, incontrast, 1+ score shows a compromised endothelial layer.

TABLE 1 Cell viability scores for the tested storage solutions. Solution60 min 90 min 120 min 150 min 180 min 240 min 300 min 1440 min HLS + +NT NT NT NT NT NT Blood + + NT NT NT NT NT NT RPMI/M199 + + NT NT NT NTNT NT HBSS +++ ++++ +++ +++ +++ + + NT GALA ++++ ++++ ++++ ++++ ++++++++ ++++ ++++ Scoring system: + 0-25% viable, ++ 26-50% viable, +++51-75% viable, ++++ 76-100% viable. Each viability score is the meanfrom at least two different patients. HLS = heparin + lidocaine +saline, Blood = blood with added heparin (50 Units/mL), RPMI/M199 =culture media in 1:1 ratio, HBSS = Hanks' balanced salt solution, GALA =HBSS with ascorbic acid, glutathione, L-arginine, and heparin. NT = nottested secondary to lack of predicted viability.

For NO measurements, typically the lumenal endothelium was identified ina field of view at 40× magnification after XYZ scanning. Bradykininstimulated egos activity in the endothelium was measured by quantitatingthe increase in DAF fluorescence due to generation of NO over 20 min at21° C. More particularly, the generation of endothelial NO intactvessels was determined using the NO indicator dye DAF-2. In this method,vessel segments were loaded with the membrane permeate diacetate form of4,5-diaminofluorescein, which is cleaved by cellular esterases to amembrane impermeant form. This dye then combines with intracellularlygenerated NO to yield the brightly fluorescent triazolofluoresceinderivative. Vessel segments were incubated with 15 μM DAF-2/DA in HBSSfor 60 minutes at 37° C. After the incubation, vessels were washed withthree changes of HBSS to remove the excess dye. The chamber bearing thesegment in 100 μl HBSS was mounted on the microscope and imaged asdescribed below. The vessel egos activity was stimulated by gentlyadding 5 μl of a 200 μM stock solution of bradykinin in HBSS, to a finalconcentration of 10 μM to the chamber. The specificity of egos activityin the vessels was measured in the presence or absence ofN^(w)-nitro-L-arginine (L-NNA). Vessel segments were pre-incubated with100 μM of L-NNA in 1.5 ml of HBSS for 30 min at 37° C., prior to furtherincubation with DAF. Basal activity of egos was measured in absence ofbradykinin stimulation, by sham incubating the vessel in HBSS forsimilar period. The Vessel segments were imaged and the nitric oxidegeneration was measured by quantitative epifluorescence multi-photonmicroscopy as described below. Temporal changes in DAF-2 fluorescencewere recorded in real time before, and 10 minutes and 20 minutes afterbradykinin treatment. Boundaries were drawn along the endothelium in 2-3regions of the vessel lumen and changes in the integrated fluorescenceintensity within each boundary was monitored over time and integratedover all pixels within the boundary for that region using BioRad LaserSharp software. Because the size and shape of the regions varied withthe source and size of the vessel, and the endothelial layer, and toeliminate effects due to variation in DAF dye loading, fluorescenceintensities from each image were normalized by those from a referenceimage recorded prior to the bradykinin treatment for each experiment.The data is expressed as temporal change in relative fluorescenceintensity, and is the average of at least three blinded experimentsperformed on different days. HLS, blood, and culture media provided verypoor protection to the endothelial cells resulting in low cellviability. HBSS was found to maintain cell viability up to 3 hours ofstorage, but was inadequate after that point. Again, in sharp contrastto the other solutions, GALA was able to maintain cell viability evenafter 1440 minutes (24 hours) of storage.

FIG. 3 depicts a comparison between HBSS and GALA solution following 5hours of storage. At two hours, no difference in cell viability can beseen. However after three hours, a marked contrast exist in terms ofcell protection between these solutions. Note in the HBSS imagesfollowing 3 and 5 hours of storage, how the endothelial layer iscompletely non-viable and has lost its architecture. The images for theGALA solution not only show near-total cell viability, but alsodemonstrate preservation of the cellular architecture. In FIG. 4, thenear-total preservation of the vein is noted after 24 hours of storagein GALA solution.

EXAMPLE 3

In order to elucidate the molecular mechanisms that are affected by thestorage conditions, the vessel conduits were stored in HLS and GALA for1-3 hours. Segments were then labeled with anti caveolin and anti egosantibodies and appropriate fluorescence tagged secondary antibodies. Theimmunofluorescently labeled vessels were imaged using the multi-photonimaging system. As shown in FIG. 5, both the caveolin and egos wererobustly labeled with the antibodies in vessels stored in GALA for 3hours. Furthermore, caveolin and egos colocalized on the plasma membraneof the EC. In contrast, these molecules were poorly labeled and did notcolocalize in vessels stored in HLS. These results clearly demonstratethat GALA protected vessel endothelium at the molecular level, ascavolin and egos were maintained at their respective functional locationseen in normal vessels. In contrast, HLS translocated these molecules tothe nonfunctional interior regions of the EC and hence did not labelwell. It is also possible that HLS caused sloughing of the EC layer andpoor labeling of these molecules. Thus long term protective effect ofGALA is clearly evident.

EXAMPLE 4

One embodiment of a perfusion device is set out in FIG. 6. Thisperfusion device 100 comprises a tissue perfusion chamber 124 in fluidiccommunication with a perfusion circuit 123 that is operably linked to apump 101 wherein said pump is regulated by a pressure transducer 104integrated into said perfusion circuit 123.

The perfusion chamber 124 defines a reservoir which may confine fluid,comprising a chamber floor 111, by a chamber side 112, with a removablechamber lid 113, said chamber side 112 having a first inlet port 114 anda second outlet port 115. Said first inlet port 114 provides anaperature through which the perfusion circuit 123 is operably linkedwith an adjustable serrated inlet nozzle 116 such that an efflux port119, operably connected to a efflux port valve 120 (in fluidiccommunication with the interior 127 of said tissue perfusion chamber124), is disposed between said inlet port 114 and said serrated inletnozzle 116.

Said second outlet port 115 provides an aperature through which theperfusion circuit 123 is operably linked with an adjustable serratedoutlet nozzle 117 such that an uptake port 102 (said uptake port havinga lumen greater than said efflux port) is operably connected to a uptakeport valve 103 (in fluidic communication with the interior 127 of saidtissue perfusion chamber 124), is disposed between said uptake port 114and said serrated outlet nozzle 117.

The present device provides for the connection of a blood vessel 105wherein the first end 130 of a blood vessel 105 is fitted over theadjustable serrated inlet valve 116 such that said first vessel end 130is secured about said adjustable serrated valve 116 with a clampingmeans 118 a and said second blood vessel end 131 of blood vessel 105 isfitted over the adjustable serrated outlet valve 117 wherein said secondvessel end 131 is secured about said adjustable serrated outlet valve117 with a clamping means 118 b. The blood vessel in the present exampleis a saphenous vein having intra luminal valves 129, associated with thetunica intima 107, that are oriented such that said valves 129 remainopen when the physiological solution 121 is circulated, in the directionindicated by the arrow 133 from the adjustable serrated inlet nozzle 116to the adjustable serrated outlet nozzle 117.

Furthermore, the blood vessel 105 may be supported according to avariety of means including, but not limited to, a mesh hammock 108suspended between said adjustable serrated inlet nozzle 116 and saidadjustable serrated outlet nozzle 117; a mesh table 109 contacted to thechamber floor 111; and convolutions 110 pre-formed in the chamber floorwhich form a corrugated plane below the blood vessel 105.

The physiological solution 121 may be added to the present deviceaccording to a variety of means. The chamber lid 113 may be removed andthe solution may be poured directly into the chamber. A reservoir ofphysiological solution 121 may be placed in fluidic communication withthe perfusion circuit 123 such that the pump 101 will draw physiologicalsolution 121 from the reservoir 134 into the perfusion device 101. Inthe alternative powdered, lyophilized, or tableted physiologicalcomponents (including GALA) may be pre-measured into the interior of theperfusion chamber such that a physiological solution is created upon theaddition of a given volume of water. Finally, the injection port 122provides direct access to the chamber interior such that the bloodvessel may be exposed to various compounds.

EXAMPLE 5

The device described in Example 4, and set out in FIG. 6, may bereconfigured according to a variety of modification including, but notlimited to, the following alterations. FIG. 7 shows how the inlet 116and outlet nozzles 117 (as shown in FIG. 6) may be divided into two ormore manifolds, thereby forming a plurality of inlet nozzles (e.g., 216a, 216 b, and 216 c) and outlet nozzles (e.g. 217 a, 217 b, and 217 c)such that a plurality of vessels (e.g. 205 a, 205 b, and 205 c) may beperfused simultaneously. In addition, the perfusion chamber 224 may beplaced on a temperature control unit 226 which allows for the heating orcooling of the circulating physiological solution 221.

EXAMPLE 6

In a preferred embodiment, the apparatus described in FIG. 6 and FIG. 7consists of a sterile, self-contained, disposable tissue perfusionchamber 124 of surgically compatible material (plastic) containing theGALA vessel preservation solution (e.g. a physiological solution). Theentire unit can be fitted on a tray on top of a pushcart in theoperating room. Specifically, a 30 cm×15 cm×15 cm disposable plasticperfusion chamber 124 with a lid 113 and an independent temperaturecontrol element 126 to control the temperature of the system. Inlet 116and outlet 117 ports that are connected to flexible tubing is threadedinto a variety of pumps 101 (e.g. pulsatile, intermittent, reversible,able to generate sine and/or square waves).

A sterile inert screen/mesh/harnmock 108 (nylon/nitrocellulose)suspended from the inlet/outlet ports support/suspend attached humanblood vessels 105. A traumatic vascular clamps 118 a are used attach thevessel(s) 105 to the nozzle(s) 116 and vascular clamps 118 b are usedattach the vessel(s) 105 to the nozzle(s) 117. A univalve(unidirectional) injection port 122 is disposed in the flexible tubing123 for injecting pharmacological agents.

A pressure transducer 104 attached to the outlet port and connected tothe pump. GALA is pumped through the system at a desired pressureregulated by an electronic feedback control loop with the pressuretransducer. The chamber 124 and the tubing 123 are filled with GALAsolution. Air pockets are thus avoided.

A larger bore uptake port 104 (on the outlet) and a smaller bore GALAefflux port 119 (on the inlet) provide continuous circulation of freshGALA from the chamber through the vessel. The bore size differentialcreate enough turbulence in the chamber so that the suspended vesselscan be continually levaged with GALA to remove any boundary layer freeradicals, and metabolites on the external surface of the vessel (e.g.the tunica adventitia 106).

EXAMPLE 7

A surgeon harvests a saphenous vein from the leg of an anesthetizedpatient and divides the vein into segments of desired length. Thesegments are transferred to the perfusion chamber 124, suspended on themesh hammock 108 submerged in GALA solution, and are clamped onto theinlet nozzles 116 with a traumatic tissue clamps. The pump 101 is runfor a short time to remove any air trapped in the vessel. The other endsof the vessel are clamped on outlet nozzles 117. The vessel is thenperfused with GALA by continuously cycling the pump 101. Any leakingbranches on the blood vessel 105 can be tied off.

In a preferred embodiment, the pump output is adjusted to maintainoptimal intra-lumenal pressure (generally 90-120 mm Hg). In addition,the uptake port valve 102 and efflux port valve 120 may be regulated toadjust the intra-lumenal pressure for a constant pump output rate. Thetemperature of the circulating physiological solution 121 may beregulated by the temperature control unit. The vessels are perfuseduntill anastomosis. The perfusion activates flow induced release ofnitric oxide and prostacychn from the endothelial cells, and thus helpdilate the vessel and also maintain its patency during storage. Furtherhigh-pressure dilation of the vessel is not required for anastomosis.The veins are also preconditioned for arterial flow and pressure.Perfusion mediated inhibition of release of vasoconstrictors andneutralization of free radicals by the GALA protects the vessel instorage during CABG surgery.

EXAMPLE 8

Distension of vein grafts prior to anastomosis is a common practice inCABG surgery. This process allows the surgeon to check for the patencyof the graft, as well as leakage. However, pressurization of the vesselabove physiological pressures with saline solutions causes aconsiderable amount of damage to the endothelium, intima and the mediaof the vessel. Using calcein-ethidium homodimer assays and multiphotonmicroscopy the detrimental effects of distension on vessel structure andfunction are easily observed. The convoluted viable endothelial regionsof the freshly excised saphenous vein, identified by the green livingcell fluorescence, were denuded and structurally damaged due todistension prior to anastomosis apparent from the considerable amount ofred fluorescence observed in the intima and media of these distendedvessels. Data not shown.

This complication is circumvented by physiological pre-conditioning anddistension, provided by the perfusion techniques of the presentinvention, that maintains structure, function, and patency of bloodvessels (especially saphenous veins) in storage during CABG surgery. Incontrast to the conventionally distended saphenous vein (300 mm Hgpressure), a robust green fluorescence of living cells was observed inthe endothelial region of saphenous vein distended by using the newlydeveloped perfusion system (90 mm Hg pressure).

EXAMPLE 9

Conventional blood vessel physiological distension techniques adverselyeffect endothelial function in these vessels. Specifically, generationof nitric oxide was completely attenuated in the conventionallydistended vessels as seen in bar graph “B” of FIG. 8 (expressed in termsof arbitrary units of normalized fluorescence activity on the y-axis).In contrast, a robust activation of egos and production of nitric oxidewas observed in physiologically distended vessels using the perfusionsystem described in the present invention as seen in bar graph “C” ofFIG. 8 (once again, expressed in terms of arbitrary units of normalizedfluorescence activity on the y-axis; with bar graph A of FIG. 8 servingas an undistended control).

In view of these data, the perfusion methods of the present inventionmaintain the structural and functional viability of blood vessels(especially saphenous veins) used as bypass thereby promoting long-termpatency of the grafts.

It is evident from the above that the present invention provides a noveland nonobvious composition and methods for the preservation of tissuesand organs both ex vivo and in situ.

1. A device comprising, an incomplete, hollow circuit defining a liquidflow path in fluidic communication with a chamber comprising an inletport and an outlet port, said incomplete circuit comprising a pumpoperably linked to a pressure transducer, said circuit terminating atfirst and second attachment points, wherein said circuit comprises anefflux port disposed between said inlet port and said first attachmentpoint and an uptake port disposed between said outlet port and saidsecond attachment point, wherein the bore of said uptake port bore islarger than the bore of said efflux port thereby resulting in a boresize differential creating enough turbulence to continuously lavage ablood vessel, such that attachment of a first end of said blood vesselto said first attachment point and a second end of said blood vessel tosaid second attachment point generates a complete, hollow circuitdefining a liquid flow path.
 2. The device of claim 1, wherein saidfirst attachment point comprises a serrated nozzle.
 3. The device ofclaim 1, wherein second attachment point comprises an adjustableserrated nozzle.
 4. The device of claim 1, wherein said chamber issterile.
 5. The device of claim 1, wherein said chamber is disposable.6. The device of claim 1, wherein said incomplete, hollow circuitcomprises tubing, said tubing terminating inside said chamber at saidfirst and second attachment points.
 7. The device of claim 1, whereinsaid tubing is in fluidic communication with a reservoir.
 8. A methodfor perfusion, comprising: a) providing i) a device comprising, anincomplete, hollow circuit defining a liquid flow path in fluidiccommunication with a chamber comprising an inlet port and an outletport, said incomplete circuit comprising a pump operably linked to apressure transducer, said circuit terminating at first and secondattachment points, wherein said circuit comprises an efflux portdisposed between said inlet port and said first attachment point and anuptake port disposed between said outlet port and said second attachmentpoint, wherein the bore of said uptake port bore is larger than the boreof said efflux port thereby resulting in a bore size differentialcreating enough turbulence to continuously lavage a blood vessel; andii) a segment of a blood vessel, said blood vessel having a first endand a second end; b) attaching, in any order, said first end of saidsegment to said first attachment point and said second end of saidsegment to said second attachment point, under conditions such that acomplete, hollow circuit defining a liquid flow path is produced; and c)circulating an aqueous solution in said hollow circuit.
 9. The method ofclaim 8, wherein said aqueous solution comprises an antioxidant andL-arginine.
 10. The method of claim 9, wherein said aqueous solutionfurther comprises an anticoagulant.
 11. The method of claim 10, furthercomprising a cellular reducing agent.
 12. The method of claim 8, whereinsaid blood vessel is an isolated vein.
 13. The method of claim 12,wherein said vein is a saphenous vein.